Mitochondrial division, through the action of a conserved DRP, mediates the intracellular distribution of mitochondria in concert with transport and tethering pathways. Regulation of mitochondrial division is critical;excessive mitochondrial division is linked to numerous diseases, including neurodegeneration. Our long-term goal is to understand the mechanism and regulation of mitochondrial division and how division proteins collaborate with other pathways to distribute mitochondria and contribute to cellular homeostasis. Using a combination of structural, biochemical, genetic, and cytological approaches in yeast and mammalian cells, we will address the outstanding question of how on the molecular level division DRPs harness the GTPase cycle to divide mitochondria. Although DRPs can function as minimal machines in vitro, in cells all require additional proteins, whose mechanisms of action are not well understood. We will determine how mitochondrial division DRP effector proteins mechanistically function in yeast and mammalian cells. This will provide new insight into how they are used to integrate mitochondrial functions with cellular signaling pathways and are co-opted to regulate non-traditional DRP cellular events, such as apoptosis. We will determine how DRPs are harnessed for different activities through the analysis of the Dnm1 interacting protein, Num1, which is a cortical protein that mediates mitochondrial tethering. Our focus on Num1 will also serve to fill the gap in our understanding of the molecular basis of tethering-based distribution, which is common in cell types, such as neurons that have a functionally important population of stationary mitochondria. The basic mechanisms of mitochondrial division and distribution and their regulation are directly relevant to our understanding of the molecular basis of an increasing number of diseases, such as Parkinson's disease and diabetes and also acute pathological conditions, such as stroke and heart attack. As such, this work will pave the way for new and better therapeutic strategies for these diseases and conditions in humans. ) PUBLIC HEALTH RELEVANCE: ) Understanding the fundamental mechanism of division and its regulation is directly relevant to understanding the basis of an increasing number of diseases associated with defects in mitochondrial division, such as Alzheimer's, Parkinson's and diabetes. The links between mitochondrial division and disease and between division proteins and apoptosis makes these proteins attractive targets for new therapeutics. Thus, the proposed research is relevant to the part of the NIH's mission that fosters fundamental basic cell biology discoveries that will directly lead the development of potentially new classes of therapeutics that target division to treat a wide array of human diseases.